A wide variety of prokaryotic and eukaryotic hosts exist and is available for the expression of heterologous genes. The features of importance for selecting an appropriate host depend on the characteristic of the protein to be produced and the applications hereof. It appears that filamentous fungi possess unique features, which make them attractive as host organisms for the production of heterologous gene products. It is furthermore known by a person of skill that certain fungal species are capable of secreting large quantities of proteins in submerged cultures. In 1991, selected strains of Aspergillus niger can produce greater than 20 g of glucoamylase per liter in industrial fermentations. During the last decade, the use of filamentous fungi for the expression and secretion of heterologous proteins has been extensively explored. For example, Berka et al. (1991) disclosed the use of strains of Aspergillus niger var. awamori as hosts for the expression and secretion of bovine chymosin and Rhizomucor miehei aspartyl protease (RmAP), two milk-clotting enzymes used commercially in cheese manufacturing to obtain coagulation (alias clotting) of the milk.
Enzymatic coagulation of milk by milk-clotting enzymes, such as chymosin and pepsin, is obviously one of the most important processes in the manufacture of cheeses. Enzymatic milk coagulation is a two-phase process: a first phase where a proteolytic enzyme, preferably an aspartic protease such as e.g. chymosin or pepsin, attacks κ-casein, resulting in a metastable state of the casein micelle structure and a second phase, where the milk subsequently coagulates and forms a coagulum.
Chymosin (EC 3.4.23.4) and pepsin (EC 3.4.23.1), the milk clotting enzymes of the mammalian stomach, are aspartic proteases that are produced naturally in the gastric mucosal cells of several mammalian species including ruminant species, porcine species, primate species and ungulate species. When produced in the gastric mucosal cells, chymosin occurs as enzymatically inactive pre-prochymosin. When chymosin is excreted, an N-terminal peptide fragment, the pre-fragment (signal peptide) is cleaved off to give prochymosin including a pro-fragment. Prochymosin is a substantially inactive form of the enzyme, which however, becomes activated under acidic conditions to the active chymosin by autocatalytic removal of the pro-fragment. The active form is termed the mature form. This activation occurs in vivo in the gastric lumen under appropriate pH conditions or in vitro under acidic conditions.
An insufficient availability of calf stomachs often occurs and a consequence of this is that the price of calf chymosin becomes subject to undesired fluctuations. For these reasons expression of bovine chymosin in microorganisms is very attractive as production method, as it will tend to hamper price level fluctuations and to date bovine chymosin cDNA sequences have been successfully expressed in bacteria, yeast and filamentous fungi.
However, major barriers for achieving higher yields of aspartic proteases such as e.g. chymosin from filamentous fungi unfortunately exist. Examples hereof include proteolytic degradation of the protein by endogenous host aspartyl proteases and inefficient secretion of the heterologous protein product from the fungi.
In order to avoid some of the above barriers, the general practice for production of non-homologous proteins in fungi is to make a fusion of the protein of interest to a highly secreted fungal carrier molecule such as e.g. glucoamylases, or amylases or cellulases. A specific cleavage site is usually introduced between the carrier molecule and the non-fungal protein. One example of such a production system is the production of chymosin by Aspergillus niger var. awamori, in which the prochymosin gene is fused to the fungal carrier glucoamylase (Ward et al., 1990).
The chymosin molecule is well characterised in the prior art. It appears that Chymosin consists of one single chain of 223 amino acids having three disulphide bridges and a molecular weight of approx. 35.000. The amino acid sequence is known. Chymosin exists in at least two iso-forms, viz. A and B. The A-form possesses the amino acid Asp in position 244, whilst the B-form has instead a Gly in the same position.
Chymosin is furthermore featured by having two N—X—S glycosylation sites which however are poorly glycosylated. The degree of glycosylation is shown to be less than 1%, when chymosin is produced in a bovine animal versus a degree of about 10% when produced by fermentation of a genetically modified Aspergillus niger var. awamori 
All aspartic proteases consist of two similar domains packed in such a manner that a deep active site cleft is formed. It appears that the amino acid Asp (nos 34 and 216, when reffering to chymosin) are the main amino acids participating in the catalysis, but also Tyr75, situated on a loop, the so-called flap, seems to influence the activity of the enzymes (Gilliland et al., 1990)
In the prior art there are conflicting opinions on whether or not the glycosylation of heterologous proteins improves the secretion of the protein from the host. Although most prior art may seem to link an improved secretion with a glycosylation of the protein (Berka et al., 1991), some authors state that the protein glycosylation may not be a prerequisite for obtaining an enhanced glycoprotein production and/or secretion from the host (Wallis et al. 1999).
According to current prior art the basic glycosylation involves the attachment of oligosaccharides to Asn (Jenkins and Curling, 1994), Ser and Thr residues in the consensus sequence Asn-X-Ser/Thr on the surface of the molecules. The oligosaccharides attached to Asn residues are referred to as N-linked, whereas those attached to Ser and Thr are designated as O-linked oligosacchaides. The N-linked glycans, which in general is to be understood as monosaccharides linked together by glycosidic bonds, have in the present context a core region of two N-acetylglucosamine residues, which provide the linkage to the protein, joined with eight mannose residues. From a review of protein secretion (Peberdy, 1994) it appears that the O-linked glycosylation of a protein is essential for the secretion, whilst the N-linked oligosaccharides appears to be of importance when providing the protein with stability and resistance to environmental influence.
Thr at position 3 in the consensus sequence Asn-X-Ser/Thr seems to lead to an increased chance of glycosylation compared to Ser at this position (Jenkins and Curling, 1994). Furthermore, several other factors may be important for the glycosylation, i.e. the position (susceptibility) of the Asn in the three-dimensional structure and different production organisms glycosylate differently (Harboe, 1998).
It appears from the prior art that it has been suggested to improve secretion of heterologous proteins produced in filamentous fungi by means of an introduction of glycosylation sites. By way of example Berka et al. (1991) demonstrate in a study that the chymosin coding region was modified by introduction of a consensus N-linked N—X—S glycosylation site (Ser74→Asn74, His76→Ser76) in a chymosin molecule that had a very low glycosylation of the two potential glycosylation sites on chymosin and no fusion partner. It was found that the production yields of extracellular chymosin were increased at least three-fold compared with the parental native chymosin having only two poorly glycosylated sites. However, it furthermore appeared that the specific activity of the so-called glycochymosin was reduced to about 20% relative to that of the native chymosin. It is evident from the wording that the experimental work was conducted in a laboratory scale and that the outcome was very low chymosin yields. One conclusion from this work is that the yield was improved from low yield to still low yield of secreted chymosin.
As the specific activity additionally dropped significantly, it must be concluded that the non-conservative substitutions conducted by Berka, are to be accorded other effects on the enzyme activity and properties.
To date, improved glycosylation has not been publicly used and described to obtain commercial levels of protein production, assumingly because it has been observed and established as an undisputed fact that increased enzyme production due to glycosylation was accompanied by severely decreased specific enzyme activity, making this a non-attractive production method.
Hitherto it appears from the prior art that no method has been disclosed whereby the production capacity of the enzyme activity is increased in this context by alteration of the glycosylation of aspartic protease such as e.g. chymosin without influencing the enzyme properties.